Clarification of blood serum and plasma using block copolymers of ethylene oxide and polyoxypropylene

ABSTRACT

A method of clarifying blood serum and plasma to remove undesired proteinaceous and lipid particulate matter which comprises admixing the serum or plasma with from greater than about 20 percent to about 30 percent of block copolymers of ethylene oxide and polyoxypropylene polymer.

United States Patent 11 1 Fekete et al.

Peetoom, Laguna Beach, both of Calif.

Baxter Laboratories, Inc., Morton Grove, Ill.

Filed: Apr. 5, 1973 Appl. No.: 348,462

Related US. Application Data Division of Ser. No. 158,111, June 29, 1971, Pat. No. 3,770,631.

Assignee:

US. Cl 260/112 B, 210/010. 23, 210/53,

424/101 Int. Cl. C02b 1/20, A23j 1/06, C07g 7/00 Field of Search 210/53,"54, DIG. 23; 424/11, l01;260/112B Oct. 1, 1974 [56] References Cited I UNITED STATES PATENTS 2,532,348 12/1950 Gyorgyi et al. 424/10] 2,533,004 12/1950 Ferry et al. 424/101 3,395,210 7/1968 Lenaban et al., 424/11 3,450,502 6/1969 Hymes 424/101 3,522,148 7/1970 Adam et al. 424/101 Primary ExaminerI-Ioward E. Schain Attorney, Agent, or FirmScott J. Meyer with from greater than about 20. percent to about 30v percent of block copolymers of ethylene oxide and polyoxypropylene polymer.

26151153, No na'wng A CLARIFICATION OF BLOOD SERUM AND PLASMA USING BLOCK COPOLYMERS OF ETIIYLENE OXIDE AND POLYOXYPROPYLENE This is a division, of application Ser. No. 158,111, filed June 29, 1971 now US. Pat. No. 3,770,631.

This invention relates to a method of separating proteinaceous and lipid materials from blood serum and plasma.

Blood comprises a fluid containing the red and the white blood cells and the blood platelets. The plasma, or fluid part of blood, contains about 90 percent water and percent solids. These solids consist essentially of about 7-9 percent proteins, 1 percent salts, and the remainder lipids and other substances. Freshly drawn blood clots within a few minutes. Formation of the clot is a complex process in which the protein, fibrinogen, is converted into insoluble fibrin. Blood serum is plasma from which this fibrin has been removed.

Fractionation of blood plasma and serum and the laboratory and clinical use of the separated blood components is common practice today. Among the various components separated from blood are albumin, a,- globulins, a- -globulins, B-globulins, y-globulins, fibrinogen, prothrombin, antihemophilic globulin, lipoproteins, thromboplastin, complement components, isoagglutinins, cholesterol, phosphatides, and numerous enzymes, e.g., amylase, fibrinolysin, esterase, and phosphatase. Various methods have been developed heretofore for separating and purifying the foregoing and other blood components. These methods generally comprise one or more of the following procedures:

a. fractional precipitation with ammonium sulfate and similar salts;

b. organic solvent precipitation with cold ethanol or acetone and other such alcohols and ketones;

c. selective adsorption on calcium phosphate gels or with barium sulfate;

d. isoelectric precipitation by pH adjustment to the point at which there is no net charge on a given protein; and

e. chromatography by use of adsorbents such as CM- or DEAE-cellulose or by Sephadex gel filtration.

Other more recently developed procedures for selectively fractionating and purifying blood proteins involve the use of amino acids such as glycine and beta alanine, water-soluble organic polymers such as polyethylene glycol and polypropylene glycol, and waterinsoluble polyelectrolyte polymers containing basic amino groups such as the dimethylaminopropylimide group.

In accordance with the present invention, a new and improved method is provided for separating proteinaceous and lipid materials from blood serum and plasma. The method comprises selective precipitation with certain block copolymers which are ethylene oxide-propylene glycol condensation products. Separation of the blood components with these block copolymers has been found to be substantially and significantly better than with the polymeric polyethylene glycol. These improvements consist of increased yield and higher purity of the precipitated protein substances, greater clarity and stability of the resulting supernatant liquid serum products, and more rapid separation of the desired components.

The ethylene oxide-propylene glycol condensation products employed in this invention can be prepared by condensing ethylene oxide with polyoxypropylene polymer. A further description of the preparation of these block copolymers is found in US. Pat. No. 2,674,619. These block copolymers can be represented by the following structural formula:

HO(CH2CH20) ,(EJHCHzO) For purposes of this invention, these block copolymers desirably contain at least 50 percent ethylene oxide in the molecule and a polyoxypropylene hydrophobic base molecular weight of at least 950. Materials containing less than 50 percent ethylene oxide are not sufficiently non-toxic and products having a hydrophobic base molecular weight less than 950 do not have the desired solubility. In this respect, the block copolymers employed in this invention are related to and include materials used as blood plasma substitutes and for priming heart-lung apparatus as described in Belgian Pat. No. 721,138 and US. Pat. Nos. 3,450,502 and 3,577,522. Said materials are described in US. Pat. I19; 2459:5 22 a -s yq na hav tbs rm 2 4 )b( 3H6O) 1(C2H4O)bH wherein a is an integer such that the hydrophobe base represented by (C H6O) has a molecular weight of at least 950 and b is an integer such that the hydrophile portion represented by (C H4O) constitutes from about 50 percent to 90 percent by weight of the compound.

' Illustrative examples of suitable block copolymers are the F-38 and F-68 PLURONIC polyols sold by Wyandotte Chemicals Corp. F-38 contains percent of polyoxyethylene hydrophilic units in the molecule and the polyoxypropylene hydrophobic base has a molecular weight of 950. F-68 also contains 80 percent of polyoxyethylene hydrophilic units in the molecule but the hydrophobic base has a molecular weight of 1,750. The total molecular weight of these two PLURONIC polyols is 4,750 and 8,750, respectively. A further description of these polyols is found in the bulletin of Wyandotte Chemicals Corp. The Pluronic Grid, Sixth Edition.

The amount of block copolymer employed in the separation process of this invention can vary, depending in part on the particular proteinaceous or lipid fraction to be precipitated from the blood plasma or serum. In general, from about 1 percent to about 30 percent of the copolymer on a basis of weight per volume of the plasma or serum is employed. Relatively low levels of the block copolymer, for example, from about 1 percent to about 20 percent are generally employed for fractionating specific protein materials which are desired to be retained for laboratory and clinical use. Higher levels of the block copolymer, for example, from greater than about 20 percent to about 30 percent, are preferred for removing substantially all undesired proteinaceous and lipid particulate matter from the blood plasma or serum. This is achieved by precipitating a product at this concentration of copolymer which can be separated from said particulate matter, which is soluble and remains in the supernatant. The precipitate can then be redissolved to form a crystal clear serum.

The precipitation can be conducted at ordinary room temperature (about 25 C) but colder temperatures are generally preferred since proteinaceous materials are known to be subject to heat denaturation and optimum potency is retained in the proteinaceous material by maintaining cold temperatures of from about 4 C to about 25 C.

In some instances it is useful to employ the block copolymer agents in combination with certain other substances which are essentially precipitation or absorption agents. In particular, glycine, tricalcium phosphate and diatomaceous earth are frequently advantageously employed in conjunction with the block copolymers. The step in the separation process at which these other agents are employed and the amounts used will vary, depending in part on the particular proteinaceous or lipid material desired to be precipitated. Generally, from about 0.5 to 2 percent of the tricalcium phosphate, about 0.1 to 0.5 percent of the diatomaceous earth, and about 2 to 3 molar glycine are suitable in these instances.

The following specific examples will illustrate the invention as it applies in particular to the preparation of antihemophilic globulin and prothrombin coagulation factors, to thromboplastin controls, and to the preparation of clarified serums for organ perfusates and for use as blood grouping and typing serums. It will be appreciated that other examples will be apparent to those skilled in the art and the invention is not limited to these specific illustrative examples.

EXAMPLE 1 The preparation of plasma for use as an organ perfusion fluid. Plasma that has been collected in anticoagulant is first treated by admixing with glycine to a molarity of 2 to 3 respective of glycine at pH 6.88 in the cold (8 C) for 30 to 45 minutes. The precipitate that forms is discarded and the remaining supernatant fluid is diluted 1:1 with normal saline (0.85 percent NaCl solution). From above 20 percent to about 30 percent (wt./vol.) of PLURONIC F-38 is then added at pH 6.5 to 7.0 and the mixture stirred for 30 minutes at room temperature. The supernatant fluid is discarded and the remaining precipitate dissolved in normal saline solution to a level of approximately the original plasma volume. To this solution is added 0.5 percent (wt./vol.) of tricalcium phosphate at pH 7.2. The suspension is mixed for 60 minutes at room temperature (25 C). The precipitate that is formed is discarded and the supernatant fluid is retained. To the supernatant fluid is added sodium citrate. to a concentration of 0.02 M. The pH is adjusted to 7.5 and the fluid is then heat treated at 37 C for 24 to 120 hours until the level of coagulation Factor V (proaccelerin) is decreased to approximately zero percent. Then 0.15 percent (wt/vol.) CELITE diatomaceous earth filter aid is added and the fluid mixed for 10 minutes at room temperature. The resulting precipitate is discarded and the remaining supernatant fluid is sterile filtered through a M1LLlPOREfilter (0.3 microns). The retained fluid is stable in storage and is crystal clear in appearance. It is eminently suitable for use as an organ perfusion fluid, for example, a kidney, heart or lung perfusion fluid.

1n the above Example, the glycine precipitation step removes essentially all the Factor V111 and fibrinogen, the PLURONlC F-38 precipitation step removes essentially all the lipoprotein and residual fibrinogen, the tricalcium phosphate absorption step removes Factors EXAMPLE 2 The preparation of serum for use as blood grouping and typing serums. The serum to be clarified is first diluted with normal saline (0.85 percent NaCl solution) so that the protein concentration is between about 2.5

and 3.0 grams/100 ml. The pH is adjusted to 6.57.0 and then from above 20 percent to about 30 percent of PLURONIC F-38 (preferably about 24 grams/ ml.) is added at room temperature and mixed for 30 to 60 minutes. The supernatant, which contains lipoprotein and fibrous materials, is discarded and to the remaining precipitate is added sufficient normal saline to adjust the volume to a level of about the original serum volume. The pH is adjusted to 7.2 and then 0.5 percent (wt./vo1.) of tricalcium phosphate is added to the dissolved precipitate. The suspension is mixed for 30 minutes at room temperature. The mixture is then centrifuged to clarify the serum and the resulting supernatant serum is carefully decanted from the tricalcium phosphate and activated clotting factors complex and retained for blood banking use. This procedure is suitable, for example, for the clarification of (a) Anti-A and Anti-B blood grouping serums, and (b) Anti-c and Anti-e typing serums for the saline tube test. In accordance with one aspect of the invention, the blood grouping and typing serum is obtained from blood serum by admixing said blood serum in aqueous dilution at a protein concentration of from about 2.5 to 3.0 grams per ml. with the block copolymer to a concentration of from greater than about 20 percent to about 30 percent on a weight per volume basis of said block copolymer, suspending the resulting precipitate by aqueous dilution to about the original serum volume, admixing the resulting suspension with from about 0.5 percent to about 2 percent tricalcium phosphate, separating the resulting precipitate therefrom and retaining the clear supernatant fluid.

EXAMPLE 3 The preparation of an AHF concentrate for clinical use. To prepare a potent antihemophilic factor (AHF, Factor V111) concentrate by fractionation with PLU- RONlC F-38, the following procedure is followed:

Whole blood is collected in 4 percent citrate anticoagulant solution (1 part of 4 percent citrate anticoagulant solution plus nine parts of whole blood); the cells are separated from the plasma by centrifugation; the retained plasma is frozen at 25 C and then thawed at 45 C; and the resulting cryoprecipitate is collected by centrifugation.

The cryoprecipitate is dissolved in 0.1 molar glycinecitrated saline (one part 0.1M sodium citrate in four parts by weight 0.9 percent saline made 0.1M respective of glycine) at 2225 C (room temperature) and the pH is adjusted to 6.5 with 1 normal acetic acid. Solid PLURONIC F-38, 3.5 percent (wt./vol.), is added and the suspension mixed for 15 minutes at 22-25 C. The mixture is centrifuged for 30 minutes at 5,000 RPM and the resulting precipitate (fibrinogen) is discarded.

The AHFrich supernatant is collected and the pH is adjusted to 6.88 with 1N NaOH. Solid PLURONIC F-38 is then added to a final concentration of 10 percent (wt/vol.) and the suspension is mixed for 15 minutes at 2225 C. The mixture is centrifuged for 30 minutes at 5,000 RPM and the supernatant is discarded.

The remaining AHF precipitate obtained by the second centrifugation is dissolved in citrated saline (one part of 0. 1M sodium citrate in four parts by weight 0.9 percent saline) to a final potency of 20 to 30 AHF units per ml. As used herein, an AHF unit is equivalent to the AHF activity in one ml. of pooled normal whole plasma. The dissolved product is sterile filtered with a MILLIPORE membrane filter using pore sizes of 0.3a, 0.45u, and 02 The filtered solution is then filled under asceptic conditions in ml. to 30 ml. capacity vials. rapidly frozen and lyophilized. Upon reconstitution with sterile water, the final product is suitable for administration to hemophiliac patients suffering bleeding episodes.

EXAMPLE 4 The preparation of a prothrombin complex for clinical use. To prepare a potent prothrombin complex (Factor ll plus other clotting factors) by fractionation with PLURONIC F-38, the following procedure is followed:

- Cohn plasma fraction lV-l is suspended in normal saline (0.9 NaCl solution) to a concentration of 10 percent (wt./vol.) and the pH is adjusted to 7.2. Tribasic calcium phosphate (500 grams) is then added to 5 0 liters of the fraction lV-l suspension and the mixture is stirred for about 30 minutes. The suspension is then centrifuged and the supernatant is discarded. The retained precipitate is suspended in 0.1M trisodium citrate to a final volume of 5 liters. The suspension is centrifuged and the precipitate is discarded. The pH of the retained supernatant is adjusted to 7.2, PLURONIC F-38 is added to a final concentration of 5 percent, and the suspension stirred for about minutes. The suspension is clarified by centrifugation, with retention of the supernatant and discarding of the precipitate. The pH of the retained supernatant is then adjusted to 5.2, and PLURONIC F-38 is added to a final concentration of percent. The suspension is centrifuged and the precipitate that is recovered is dissolved in citrated saline to a volume of 5 liters. Heparin is added in an amount of one unit per ml., and the dissolved product is sterile filtered with a MILLIPORE membrane filter using pore sizes of 0.3a, 0.45p., and 0.2,u. The filtered solution is then filled under aseptic conditions in 10 ml. to ml. capacity vials, rapidly frozen and lyophilized. Upon reconstitution with sterile water, the final product is suitable for administration to patients deficient in Factor II.

In Examples 3 and 4, above, it is found that the separation of the respective desired protein precipitate is achieved more rapidly than is the case when polyethylene glycol 4,000 is used in place of the PLURONIC F-38 and the yield of the final product is substantially increased.

EXAMPLE 5 The preparation of a thromboplastin control from plasma. Fresh human blood is collected in 4 percent sodium citrate anticoagulant solution (one part of anticoagulant and nine parts of whole blood). The resulting mixture is centrifuged at 3,000 X G at 5 C. The resulting supernatant plasma is centrifuged again in the same manner as before and then frozen at -20 C. The plasma is thawed at room temperature (ca. 25 C) and diluted with 0.9 percent physiologically normal saline solution (one part of plasma and one part of normal saline solution). The pH of the resulting solution is adjusted to 6.07.0 with 1N HCl. PLURONIC F323 is then added to the solution to a final concentration of 30 percent to form a precipitate. After centrifugation,

excess citrate and trace amounts of thromboplastic active material are removed in the supernatant, which is discarded.

The above-prepared plasma is then used to prepare several thromboplastin controls for use in making prothrombin time determinations such as by the Quick one-stage prothrombin time test or the modified Owren prothrombin test as described in the Hyland Reference Manual of Coagulation Procedures, published by Hyland Laboratories, Los Angeles, Calif, pp. 10-14 (2d ed. 1964). These controls are made to resemble physiological plasma by employing as the sole buffering agent for the plasma (1) citrate-imidazole-normal saline buffer, or (2) oxalate-imidazole-normal saline buffer, consisting of the following components:

1. Citrate-imidazole-normal saline buffer 0.9 percent NaCl solution 0.0135 molar i 0.005 tri-sodium citrate 0.025 to 0.035 percent imidazole 2. Oxalate-imidazole-normal saline buffer 0.9 percent NaCl solution p 0.0135 molar i 0.005 sodium oxalate 0.025 to 0.035 percent imidazole By using the foregoing amounts of the citrate or oxalate anticoagulants in said buffer, undesirable activation of the clotting factors and thromboplastin generation are effectively prevented. Excess citrate and traces of thromboplastic active material are removed. as hereinbefore provided, by the initial employment of the PLURONIC F-38 treatment on the plasma. With these combined steps, maximum stabilization of the thromboplastin controls is achieved.

The citrate controls (100 percent and 20 percent controls) are prepared as follows:

For the 100 percent control, the precipitant paste from the PLURONIC F-38 treatment is dissolved in the citrate-imidazole-normal saline buffer to 50 to percent of the original volume of the plasma.

For the 20 percent control, the precipitant paste from the PLURONIC F-38 treatment is dissolved in normal saline to to 120 percent of the original volume of the plasma. Prothrombin-free human plasma is then prepared from the redissolved precipitant paste by adsorbing out the prothrombin complex (Factors ll, VII, IX and X). This adsorption is achieved by adding tricalcium phosphate to a final concentration of 1 percent by weight, adjusting the pH to 7.2 i 0.2 with 1 normal HCl, and mixing for about 30 to 45 minutes. The suspension is then centrifuged. The supernatant is buffered with 0.0135M i 0.005 trisodium citrate and 0.025 to 0.035 percent imidazole, and the pH adjusted to 7.8 i 0.2 with 1 normal NaOH. This solution is then mixed with the appropriate volume of the aboveprepared percent citrate control to provide the desired clotting times for the 20 percent control.

The 100 and 20 percent oxalate controls are prepared in the same manner as the above-prepared citrate controls except that 0.0135M sodium oxalate is used in place of 0.0135M trisodium citrate.

Various other examples and modifications of the foregoing examples will be apparent to those skilled in the art. For example. albumin, y-globulins, fibrinogen and other blood fractions can also be obtained by employing the separation procedure of this invention. All such further examples and modifications as come within the spirit and scope of the invention are included herein.

What is claimed is:

1. The process in which a clarified serum for use as a blood grouping and typing serum is obtained from blood serum by admixing said blood serum in aqueous dilution at a protein concentration of from about 2.5 to 3.0 grams per 100 ml. with a compound of the formula such that the hydrophobic portion represented by has a molecular weight of at least about 950 to a concentration of from greater than about 20 percent to about 30 percent on a weight per volume basis of said compound, admixing the resulting precipitate by aqueous dilution to about the original serum volume, admixing the resulting mixture with from about 0.5 percent to about 2 percent tricalcium phosphate, separating the resulting precipitate therefrom and retaining the clear supernatant fluid.

2. The process of claim 1 in which the block copolymer contains about percent of polyoxyethylene hydrophilic units in the molecule and the polyoxypropylene hydrophobic base has a molecular weight of 950.

I UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. SL839, 314 Dated OCtOber 1974 Inv Lajos F. Fekete and Frans Peetoom It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column 4,' line 28, a separate paragraph should begin with the word "In". Column 7, line 16, the formula should appear as follows:

H0(CH CH O)% ((III-ICI-I O) (CH CH O) H CH Column 8, line 3, the formula should appear as follows: (CI-ICH O) Signed and sealed this 10th day of December 1974.

(SEAL) Attest:

McCOY M. GIBSON JR. C. MARSHALL DANN Arresting Officer I Comissioner of Patents USCOMM-DC 603764 69 FORM PC4050 (10-69) v us, covzmmzm "mums omc: m9 o-au-su 

1. THE PROCESS IN WHICH A CLARIFIED SERUM FOR USE AS A BLOOD GROUPING AND TYPING SERUM IS OBTAINED FROM BLOOD SERUM BY ADMIXING SAID BLOOD SERUM IN AQUEOUS DILUTION AT A PROTEIN CONCENTRATION OF FROM ABOUT 2.5 TO 3.0 GRAMS PER 100 ML. WITH A COMPOUND OF THE FORMULA
 2. The process of claIm 1 in which the block copolymer contains about 80 percent of polyoxyethylene hydrophilic units in the molecule and the polyoxypropylene hydrophobic base has a molecular weight of
 950. 